McCowage G B, Phillips K L, Gentry T L, Hull S, Kurtzberg J, Gilboa E, Smith C
Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710, USA.
Exp Hematol. 1998 Apr;26(4):288-98.
Retroviral vector gene transfer strategies are currently being developed to treat a variety of hematopoietic disorders. To date, genetic modification of human pluripotent hematopoietic stem cells has been inefficient. In the present study we developed reagents and procedures for rapidly screening retroviral vector gene transfer conditions using a multiparameter fluorescence-activated cell sorting (FACS) assay. To identify transduced cells using FACS analysis, we developed a retroviral vector, termed MN, which stably expressed high levels of a truncated version of the low-affinity nerve growth factor receptor (LNGFR). In addition, procedures were developed for enriching CD34+ cells from cryopreserved umbilical cord blood. These cells were transduced with MN and evaluated using multiparameter FACS analysis for expression of CD34, CD38, and LNGFR. Stem cell maintenance was determined by measuring the CD34hi and CD34hiCD38lo/- cells remaining after ex vivo gene transfer. Gene transfer into these cells was measured by evaluating cells expressing high levels of LNGFR. Initial studies with this assay and with in vitro functional assays indicated that retroviral gene transfer following pre-incubation with a variety of cytokines in serum containing conditions resulted in 1) poor maintenance of hematopoietic stem cells and 2) gene transfer predominantly in relatively mature cells. When gene transfer in serum-free conditions was performed, some improvement was observed in the maintenance of cells retaining primitive immunophenotypes with no reduction in the gene transfer efficiency. The MN vector and multiparameter FACS analysis will be useful in efficiently screening ongoing efforts designed to improve stem cell gene transfer.
目前正在开发逆转录病毒载体基因转移策略，以治疗多种造血系统疾病。迄今为止，人类多能造血干细胞的基因修饰效率一直很低。在本研究中，我们开发了试剂和程序，用于使用多参数荧光激活细胞分选（FACS）分析快速筛选逆转录病毒载体基因转移条件。为了使用FACS分析鉴定转导细胞，我们开发了一种称为MN的逆转录病毒载体，该载体稳定表达高水平的低亲和力神经生长因子受体（LNGFR）的截短版本。此外，还开发了从冷冻保存的脐带血中富集CD34+细胞的程序。这些细胞用MN转导，并使用多参数FACS分析评估CD34、CD38和LNGFR的表达。通过测量体外基因转移后剩余的CD34hi和CD34hiCD38lo/-细胞来确定干细胞的维持情况。通过评估表达高水平LNGFR的细胞来测量这些细胞中的基因转移。对该分析和体外功能分析的初步研究表明，在含血清条件下与多种细胞因子预孵育后进行逆转录病毒基因转移，会导致1）造血干细胞维持不佳，以及2）基因转移主要发生在相对成熟的细胞中。当在无血清条件下进行基因转移时，在维持保留原始免疫表型的细胞方面观察到了一些改善，且基因转移效率没有降低。MN载体和多参数FACS分析将有助于有效筛选旨在改善干细胞基因转移的正在进行的研究工作。
